We have previously demonstrated that human cells can be genetically engineered to produce intracellular antibodies "intrabodies" that bind the HIV-1 envelope glycoprotein in the ER and inhibit processing of the envelope protein, syncytium formation and production of infectious virus. We have now extended this technology to target the critically important HIV-1 regulatory protein Tat and have demonstrated that we can inhibit Tat-mediated LTR transactivation and HIV-1 replication. Accordingly, the overall goals of this project are to determine if single chain antibodies (sFvs) that recognize HIV-1 Tat can be efficiently delivered into CD4+ T cells for AIDS gene therapy. Several anti-Tat MAbs will be first epitope mapped and those MAbs that map to important known activation domains will be further engineered as sFvs (and their modified forms) for intracellular expression in eukaryotic cells. We will use of plasmid based eukaryotic expression vectors to determine the optimal promoter(s) to efficiency express the anti-Tat sFvs, in the cytoplasm of CD4+ T lymphocytic cell lines. The ability of the cytoplasmically expressed anti-Tat sFvs to bind Tat will be determined. We will also determine if the anti-Tat sFvs can inhibit Tat-mediated transactivation of HIV-1 LTR CAT activity. Stable CD4+ T cell lines expressing the anti-Tat sFvs will be established. These cell lines will be challenged with several laboratory strains of HIV-1 and inhibition of infectious virus production will be quantitated. Long term cultures will be followed for the development of HIV-1 escape mutants. To study the effect of the stably transduced anti-Tat sFv genes on inhibition of HIV-1 replication in chronically HIV-1 infected cell lines, the latently infected U1 promonocytic cell line and the T-lymphocytic cell line ACH-2 will be used. To transduce primary CD4+ T cells at a high level of efficiency for AIDS gene therapy, two different gene transfer systems will be used. For retroviral mediated gene transfer, we will use the N2 based retroviral vector and will establish high titer amphotropic packaging cell lines. Supernatants will be harvested and used to transduce the sFvtat genes at a high level of efficiency into CD4+ T cell lines as well as into uninfected and HIV-1-infected CD4+ primary lymphocytes. Inhibition of production of infectious HIV-1 virions will be examined following challenge with different laboratory strains and primary isolates of HIV-1. For non-retroviral mediated gene transfer of sFvtat genes, an adeno-associated virus (AAV) vector will be used. Encapsidated viral stocks will be generated, stable cell lines will be established and then challenged with HIV-1 as described above. Finally, to test for combined or synergistic inhibition of HIV-1 replication we will use both vectors to transduce both the anti-env and the anti-Tat sFvs into CD4+ T cell lines and primary lymphocytes and will compare the inhibition to the anti-Tat sFv alone. These experiments now addressing the effects of combined HIV-1 targets will supply valuable information for future gene therapy clinical trials.